Attachment of PC12 Cells to Adhesion Substratum Induces the Accumulation of Glucose Transporters (GLUTs) and Stimulates Glucose Metabolism

The levels of glucose transporters (GLUTs), specifically GLUT3 and GLUT1, increased dramatically in PC12 cells that were cultured on suitable adhesion substrata (poly-l-lysine [PLL]) and induced to differentiate with nerve growth factor (NGF). Closer examination of this response revealed that: (1) cellular attachment to PLL was sufficient to stimulate the increase in GLUT immunoreactivity, and (2) NGF alone was not effective unless the cells were cultured on PLL-treated surfaces. The response to PLL was detected as early as 4 hr after plating the cells and peaked within 24–48 hr. Other adhesion substrata, such as collagen and poly-l-ornithine, evoked a similar response, although the latter polymer was far less effective. The increase in GLUTs appeared to result from an accumulation of existing transporters because this response was not blocked by inhibiting protein synthesis. Cellular adhesion to PLL was also accompanied by a rapid activation of glucose metabolism. Thus, specific recognition of the adhesion substratum not only provides a context for cell attachment, but also elicits important functional changes in GLUT activity.     

Dual effect of nerve growth factor on cell death of PC12 cells Induced by serum deprivation

The effect of nerve growth factor (NGF) on the cell death of PC12 cells that is induced by serum deprivation was examined in the floating and attached cells to the extracellular matrix. NGF suppressed cell death occurred in the floating cells. The onset of cell death in the attached cells was much slower than in the floating cells. Moreover, the cell death in the attached cells was either accelerated in a high-density culture (over approximately 50% confluent), or inhibited in a low-density culture by NGF. While nucleosomal DNA fragmentation and poly (ADP-ribose) polymerase degradation was observed in both the floating and attached cells, the incidence of nuclear fragmentation and chromatin condensation was much lower in the attached cells than in the floating cells. The delayed onset of cell death in the attached cells was due to the signals that are generated from the extracellular matrix that is formed by PC12 cells, together with cell-to-cell interaction. The acceleration of cell death in the NGF-treated cells was anoikis, caused by the loss of the anchorage of the cell via the action of increased activities of matrix metalloproteinases (MMP2, MMP9). These results suggest that NGF has a different role in the cell death of PC12 cells that is induced by serum deprivation, depending on the cell-matrix, as well as the cell-cell interaction.     

Prostaglandin E1 transported into cells blocks the apoptotic signals induced by nerve growth factor deprivation

Neuronal apoptosis in rat pheochromocytoma PC12 cells, which was confirmed by TUNEL (terminal transferase-mediated dUTP-biotin nick end-labeling) staining and detection of chromatin condensation, appeared within 8 h after nerve growth factor (NGF) deprivation. Prostaglandin (PG) E1 (10(-7)-10(6) M) reduced the incidence of apoptotic cell death in PC12 cells. The genes encoding PG transporter specific to prostaglandins such as PGE2 or PGF2alpha were expressed in the cell lines as shown by RT-PCR. Bromcresol green, an inhibitor of PG transporter, reversed the antiapoptotic effect of PGE1. Moreover, treatment of PC12 cells with an antisense oligonucleotide corresponding to PG transporter cDNA also blocked the inhibitory effects of PGE1 on apoptotic cell death. In addition, PGE1 counteracted the increased activities of stress-activated protein kinase/cJun N-terminal kinase within 1-2 h after NGF deprivation in PC12 cells. These results indicated that the antiapoptotic effect of PGE1 in NGF-deprived PC12 cells was achieved by inhibitory signals following uptake into neurons through the PG transporter.     

Cyclosporin A inhibits caspase-independent death of NGF-deprived sympathetic neurons: a potential role for mitochondrial permeability transition

Opening of the permeability transition pore (PTP) has been implicated as an important mitochondrial event that occurs during apoptosis. We examined the role of the PTP in the well-characterized cell death of rat sympathetic neurons deprived of nerve growth factor (NGF) in vitro. Removal of NGF causes these neurons to undergo either a classic apoptotic cell death or, when treated with a broad-spectrum caspase inhibitor such as boc-aspartyl(OMe)-fluoromethylketone (BAF), a delayed, nonapoptotic cell death. The PTP inhibitor, cyclosporin A (CsA), blocked commitment-to-die in the presence of BAF, as defined by the ability of NGF readdition to rescue cells, but had little effect on commitment-to-die in the absence of BAF. CsA did not have trophic effects on BAF-saved cells, but did block the decrease in mitochondrial membrane potential. These data suggest that PTP opening is a critical event in caspase-independent, nonapoptotic (but not caspase-dependent, apoptotic) death of NGF-deprived rat sympathetic neurons.     

Potentiation of early necrotic death of glucose-starved pheochromocytoma 12 cells by nerve growth factor

Recently suggested is an arguable hypothesis that neurotrophins can induce necrosis but suppress apoptosis of target cells in some pathological conditions. We examined this hypothesis by tracing the type of NGF-promoted cell death occurring in a hypoglycemic condition at various angles, such as kinetic analyses, histological examinations of membrane alterations, morphological observations in ultra-structural changes, and determinations of DNA fragmentation. Glucose-starved cell death consisted of two kinetically different stages, suggesting that it be mixed with early and delayed death. Several lines of evidence revealed that NGF prominently enhanced the early death with necrotic characters. By contrast, apoptotic characters of glucose-starved delayed death were not much affected by NGF. Nifedipine, a voltage-gated calcium channel blocker, could completely compensate for the enhancement of the early glucose-starved death by NGF. Interestingly, the NGF-promoted cell death was also blocked by cycloheximide that did not keep PC12 cells alive from glucose starvation. Therefore, all the data in this study suggest that NGF accelerates the early necrosis of glucose-starved cell death probably through the alterations of intracellular calcium ions and protein syntheses.     

Apoptotic cell death in TrkA-overexpressing cells: kinetic regulation of ERK phosphorylation and caspase-7 activation

The TrkA tyrosine kinase is activated by autophosphorylation in response to NGF, and plays an important role in cell survival, differentiation, and apoptosis. To investigate its role in cell fate determination, we produced stable TrkA-inducible SK-N-MC and U2OS cell lines using the Tet-On system. Interestingly, TrkA overexpression induced substantial cell death even in the absence of NGF, by stimulating ERK phosphorylation and caspase-7 activation leading to PARP cleavage. TrkA-mediated cell death was shown by the annexin-V binding assay to be, at least in part, apoptotic in both SK-N-MC and U2OS cells. Furthermore, the truncated form (p18) of Bax accumulated in the TrkA-induced cells, suggesting that TrkA induces mitochondria-mediated apoptosis. NGF treatment augmented the cell death induced by TrkA overexpression. This TrkA-induced cell death was blocked by the tyrosine kinase inhibitors, K-252a and GW441756. Moreover, TrkA overexpression inhibited long-term proliferation of both the neuronal SK-N-MC cells and the non-neuronal U2OS cells, suggesting a potential role of TrkA as a tumor suppressor.     

Iron enhances NGF-induced neurite outgrowth in PC12 cells

Rat pheochromocytoma 12 (PC12) cells undergo neuronal differentiation in response to nerve growth factor (NGF). NGF-induced differentiation involves a number of protein kinases, including extracellular signal-regulated kinase (ERK). We studied the effect of iron on neuronal differentiation, using as model the neurite outgrowth of PC12 cells triggered by NGF when the cells are plated on collagen-coated dishes in medium containing 1% serum. The addition of iron enhanced NGF-mediated cell adhesion, spreading and neurite outgrowth. The differentiation-promoting effect of iron seems to depend on intracellular iron, since nitrilotriacetic acid (an efficient iron-uptake mediator) enhanced the response to iron. In agreement with this, intracellular, but not extracellular, iron enhanced NGF-induced neurite outgrowth in pre-spread PC12 cells, and this was correlated with increased ERK activity. Taken together, these data suggest that intracellular iron promotes NGF-stimulated differentiation of PC12 cells by increasing ERK activity.     

Sea urchin hyalin: appearance and function in development

Embryonic chicken sensory cells from dorsal root ganglia and a clonal line of pheochromocytoma cells (PC-12) extended neuronal-like processes within 24 hr of seeding on a naturally produced, basement membrane-like extracellular matrix (ECM) in the absence of nerve growth factor (NGF). Plating on ECM also induced a rapid cell attachment and flattening of these cells and supported the survival of embryonic sensory cells in primary cultures. Unlike the effect of NGF on PC-12 cells, the ECM-induced morphological differentiation was transient and led to disintegration and degeneration of processes bearing PC-12 cells. The ECM-induced morphological differentiation was not inhibited by anti-NGF antibodies, and the cells retained their ability to bind and internalize NGF in a manner similar to that observed on plastic. PC-12 cell attachment and flattening occurred on dishes coated with collagen type IV in a way similar to that observed on ECM, but precoating the dishes with fibronectin had no effect. Extension of cell processes was not induced by either substrate. Morphological differentiation but not the induction of cell adhesion and flattening was inhibited by either prefixation with glutaraldehyde, oxidation with periodate, or preexposure to concanavalin A of the ECM, suggesting that the ECM and in particular its sugar moieties play an active role in the induction of neurite outgrowth. It is suggested that close contact with the ECM provides chemical or mechanical cues that permit contactmediated elongation and directed growth of both embryonic and regenerating nerve fibers.     

Sodium nitroprusside prevents chemical hypoxia-induced cell death through iron ions stimulating the activity of the Na+-Ca2+ exchanger in C6 glioma cells

In C6 glioma cells exposed to chemical hypoxia, an increase of extracellular lactate dehydrogenase (LDH) activity, cell death, and intracellular Ca2+ concentration ([Ca2+]i) occurred. Sodium nitroprusside (SNP), a nitric oxide donor and an iron-containing molecule, reduced chemical hypoxia-induced LDH release and cell death. These effects were counteracted by bepridil and by 5-(N-4-chlorobenzyl)-2',4'-dimethylbenzamil (CB-DMB), two specific inhibitors of the Na+-Ca2+ exchanger. SNP also increased the activity of the Na+-Ca2+ exchanger as a Na+ efflux pathway, stimulated by Na+-free conditions and evaluated by monitoring [Ca2+]i in single cells. In addition, SNP produced a further increase of chemical hypoxia-elicited [Ca2+]i elevation, and this effect was blocked by bepridil. Chemical hypoxia-evoked cell death and LDH release were counteracted by the ferricyanide moiety of the SNP molecule, K3Fe(CN)6, and by ferric chloride (FeCl3), and this effect was counteracted by CB-DMB. In addition, the iron ion chelator deferoxamine reversed the protective effect exerted by SNP on cell injury. Collectively, these findings suggest that the protective effect of SNP on C6 glioma cells exposed to chemical hypoxia is due to the activation of the Na+-Ca2+ exchanger operating as a Na+ efflux-Ca2+ influx pathway induced by iron present in the SNP molecule.     

Nerve growth factor is expressed by postmitotic avian retinal horizontal cells and supports their survival during development in an autocrine mode of action

Cell death in the developing retina is regulated, but so far little is known about what factors regulate the cell death. Several neurotrophic factors and receptors, including the neurotrophins and Trk receptors, are expressed during the critical time. We have studied the developing avian retina with respect to the role of nerve growth factor (NGF) in these processes. Our starting point for the work was that NGF and its receptor TrkA are expressed in a partially overlapping pattern in the inner nuclear layer of the developing retina. Our results show that TrkA and NGF-expressing cells are postmitotic. The first NGF-expressing cells were found on the vitreal side of the central region of E5.5-E6 retina. This pattern changed and NGF-expressing cells identified as horizontal cells were later confined to the external inner nuclear layer. We show that these horizontal cells co-express TrkA and NGF, unlike a subpopulation of amacrine cells that only expresses TrkA. In contrast to the horizontal cells, which survive, the majority of the TrkA-expressing amacrine cells die during a period of cell death in the inner nuclear layer. Intraocular injections of NGF protein rescued the dying amacrine cells and injection of antisense oligonucleotides for NGF that block its synthesis, caused death among the TrkA-expressing horizontal cells, which normally would survive. Our results suggest that NGF supports the survival of TrkA expressing avian horizontal cells in an autocrine mode of action in the retina of E10-E12 chicks. The cells co-express TrkA and NGF and the role for NGF is to maintain the TrkA-expressing horizontal cells. The TrkA-expressing amacrine cells are not supported by NGF and subsequently die. In addition to the effect on survival, our results suggest that NGF plays a role in horizontal cell plasticity.     

NGF Retards apoptosis in chick embryo bursal cell in vitro

Abstract. Recent studies have demonstrated that the action of nerve growth factor (NGF) is not restricted to neuronal cells but also affects cells of the immune system. In a previous work on the effect of NGF on the chick embryo bursa of Fabricius both in vivo and in vitro, we observed that NGF prolongs bursal cell survival in vitro. In the present study we report that the increase of viable cells in NGF-treated cultures is not due to a proliferative effect of NGF on bursal cells but to a reduction of cell mortality. The morphological analysis revealed that bursal cells in cultures die by apoptosis, which was also shown by the typical pattern of DNA fragmentation, a hallmark of this cell death process. It is concluded that NGF, with an action similar to that described in sympathetic neurons and PC12, could retard bursal cell death by influencing apoptosis.     

Viral proteins E1B19K and p35 protect sympathetic neurons from cell death induced by NGF deprivation

To study molecular mechanisms underlying neuronal cell death, we have used sympathetic neurons from superior cervical ganglia which undergo programmed cell death when deprived of nerve growth factor. These neurons have been microinjected with expression vectors containing cDNAs encoding selected proteins to test their regulatory influence over cell death. Using this procedure, we have shown previously that sympathetic neurons can be protected from NGF deprivation by the protooncogene Bcl-2. We now report that the E1B19K protein from adenovirus and the p35 protein from baculovirus also rescue neurons. Other adenoviral proteins, E1A and E1B55K, have no effect on neuronal survival. E1B55K, known to block apoptosis mediated by p53 in proliferative cells, failed to rescue sympathetic neurons suggesting that p53 is not involved in neuronal death induced by NGF deprivation. E1B19K and p35 were also coinjected with Bcl-Xs which blocks Bcl-2 function in lymphoid cells. Although Bcl-Xs blocked the ability of Bcl- 2 to rescue neurons, it had no effect on survival that was dependent upon expression of E1B19K or p35.     

Magnesium-dependent attachment and neurite outgrowth by PC12 cells on collagen and laminin substrata

We report a study of the substratum and medium requirements for attachment and neurite outgrowth by cells of the pheochromocytoma-derived PC12 line. In attachment medium containing both Ca2+ and Mg2+, more than 50% of cells attached within 1 hr to petri dishes coated with native collagen Types I/III or II, native or denatured collagen Type IV, laminin, wheat germ agglutinin (WGA), or poly-L-lysine; attachment to dishes coated with nerve growth factor (NGF) was only about 20% and attachment to uncoated dishes or to dishes coated with fibronectin or gelatin was almost nil. Neither prior culturing in the presence of NGF nor addition of NGF to the attachment medium significantly affected the extent of attachment to collagen or laminin. With Ca2+ (1 mM) as the sole divalent cation, cells attached normally to WGA, polylysine, and NGF, but failed to attach to collagen or laminin. With Mg2+ (1 mM) as the only divalent cation, attachment to all substrata was about the same as in medium with both Ca2+ and Mg2+. Like the ionic requirements, the kinetics of attachment, insensitivity to protease treatment of the cells, and inhibition by low temperature and sodium azide were similar for PC12 attachment to collagen and laminin, suggesting that a common molecular mechanism may underlie attachment to these substrata. The only significant difference observed was that addition of WGA (30 micrograms/ml) to the attachment medium inhibited attachment to collagen but promoted attachment to laminin. Finally, PC12 cells extended neurites on laminin, on native collagens I/III, II, and IV, and on denatured collagen IV; they did not extend neurites on denatured collagens I/III or II, NGF, or WGA. Neurite outgrowth on collagen and laminin occurred with Mg2+ as the sole divalent cation. These results suggest that the same Mg2+-dependent adhesion mechanism operates at the cell body and at the growth cone.     

The reduced catalase expression in TrkA-induced cells leads to autophagic cell death via ROS accumulation

TrkA receptor activation is a pivotal process for neuronal cell differentiation and survival. However, its overactivation or removal of its ligand NGF tends to cause the cell death. Recently, we demonstrated that TrkA overexpression induces cell death via apoptosis. In this study we also show that the TrkA-mediated cell death is associated with autophagy. TrkA-induced cells revealed an increase of GFP-LC3 punctate formation, development of acidic vesicular organelles (AVO) and formation of autophagosomes, which were eventually blocked by the addition of some autophagy inhibitors such as 3-methyladenine, ammonium chloride or wortmannin. In addition, although expression of autophagy-related proteins such as LC3-II or Beclin-1 was subtly altered during the TrkA-mediated cell death, depletion of ATG5 or Beclin-1 substantially decreased cell death in TrkA-expressing cells. In particular, reactive oxygen species (ROS) were dramatically accumulated in TrkA-induced cells, and the high accumulation of ROS was released by treatment of autophagy inhibitors. Furthermore, addition of an antioxidant N-acetylcysteine promoted the survival of TrkA-expressing cells and suppressed AVO production in cells. We also showed that this ROS accumulation was closely associated with reduction of catalase expression. Taken together, TrkA overexpression causes ROS accumulation via reduced catalase expression, ultimately leading to autophagic cell death.     

Poly(L-lysine)-modified iron oxide nanoparticles for stem cell labeling

New surface-modified iron oxide nanoparticles were developed by precipitation of Fe(II) and Fe(III) salts with ammonium hydroxide and oxidation of the resulting magnetite with sodium hypochlorite, followed by the addition of poly( L-lysine) (PLL) solution. PLL of several molecular weights ranging from 146 ( L-lysine) to 579 000 was tested as a coating to boost the intracellular uptake of the nanoparticles. The nanoparticles were characterized by TEM, dynamic light scattering, FTIR, and ultrasonic spectrometry. TEM revealed that the particles were ca. 6 nm in diameter, while FTIR showed that their surfaces were well-coated with PLL. The interaction of PLL-modified iron oxide nanoparticles with DMEM culture medium was verified by UV-vis spectroscopy. Rat bone marrow stromal cells (rMSCs) and human mesenchymal stem cells (hMSC) were labeled with PLL-modified iron oxide nanoparticles or with Endorem (control). Optical microscopy and TEM confirmed the presence of PLL-modified iron oxide nanoparticles inside the cells. Cellular uptake was very high (more than 92%) for PLL-modified nanoparticles that were coated with PLL (molecular weight 388 00) at a concentration of 0.02 mg PLL per milliliter of colloid. The cellular uptake of PLL-modified iron oxide was facilitated by its interaction with the negatively charged cell surface and subsequent endosomolytic uptake. The relaxivity of rMSCs labeled with PLL-modified iron oxide and the amount of iron in the cells were determined. PLL-modified iron oxide-labeled rMSCs were imaged in vitro and in vivo after intracerebral grafting into the contralateral hemisphere of the adult rat brain. The implanted cells were visible on magnetic resonance (MR) images as a hypointense area at the injection site and in the lesion. In comparison with Endorem, nanoparticles modified with PLL of an optimum molecular weight demonstrated a higher efficiency of intracellular uptake by MSC cells.     

Cyclosporine A- and FK506-induced apoptosis in PC12 cells

The purpose of this study was to examine, using glycogen synthase kinase (GSK) inhibitors, whether GSK-3 is involved in cyclosporine A (CsA)- and FK506-induced apoptosis in PC12 cells. CsA and FK506 increased apoptotic cell death with morphological changes characterized by cell shrinkage and nuclear condensation or fragmentation. Nerve growth factor (NGF) completely blocked cell death. Caspase-3 activation was accompanied by CsA- and FK506-induced cell death and inhibited by NGF. GSK-3 inhibitors such as alsterpaullone and SB216763 prevented CsA- and FK506-induced apoptosis. These results suggest that CsA and FK506 induce caspase-dependent apoptosis and that GSK-3 activation is involved in CsA- and FK506-induced apoptosis in PC12 cells.     

The role of osteoprotegerin and tumor necrosis factor-related apoptosis-inducing ligand in human microvascular endothelial cell survival

Endothelial cell survival and antiapoptotic pathways, including those stimulated by extracellular matrix, are critical regulators of vasculogenesis, angiogenesis, endothelial repair, and shear-stress-induced endothelial activation. One of these pathways is mediated by alpha(v)beta(3) integrin ligation, downstream activation of nuclear factor-kappaB, and subsequent up-regulation of osteoprotegerin (OPG). In this study, the mechanism by which OPG protects endothelial cells from death was examined. Serum-starved human microvascular endothelial cells (HMECs) plated on the alpha(v)beta(3) ligand osteopontin were protected from cell death. Immunoprecipitation experiments indicated that OPG formed a complex with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in HMECs under these conditions. Furthermore, inhibitors of TRAIL, including recombinant soluble TRAIL receptors and a neutralizing antibody against TRAIL, blocked apoptosis of serum-starved HMECs plated on the nonintegrin attachment factor poly-d-lysine. Whereas TRAIL was unable to induce apoptosis in HMECs plated on osteopontin, the addition of recombinant TRAIL did increase the percentage of apoptotic HMECs plated on poly-d-lysine. This evidence indicates that OPG blocks endothelial cell apoptosis through binding TRAIL and preventing its interaction with death-inducing TRAIL-receptors     

Interaction of the 70,000-mol-wt amino-terminal fragment of fibronectin with the matrix-assembly receptor of fibroblasts

Plasma fibronectin binds saturably and reversibly to substrate-attached fibroblasts and is subsequently incorporated into the extracellular matrix (McKeown-Longo, P.J., and D. F. Mosher, 1983, J. Cell Biol., 97:466-472). We examined whether fragments of fibronectin are processed in a similar way. The amino-terminal 70,000-mol-wt catheptic D fragment of fibronectin bound reversibly to cell surfaces with the same affinity as intact fibronectin but did not become incorporated into extracellular matrix. The 70,000-mol-wt fragment blocked binding of intact fibronectin to cell surfaces and incorporation of intact fibronectin into extracellular matrix. Binding of the 70,000-mol-wt fragment to cells was partially abolished by cleavage into 27,000-mol-wt heparin-binding and 40,000-mol-wt gelatin-binding fragments and more completely abolished by reduction and alkylation of disulfide bonds. Binding of the 70,000-mol-wt fragment to cells was not blocked by gelatin or heparin. When coated onto plastic, the 70,000-mol-wt fragment did not mediate attachment and spreading of suspended fibroblasts. Conversely, fibronectin fragments that had attachment and spreading activity did not block binding of exogenous fibronectin to substrate-attached cells. These results indicate that there is a cell binding site in the 70,000-mol-wt fragment that is distinct from the previously described cell attachment site and is required for assembly of exogenous fibronectin into extracellular matrix.